Process for the preparation of dihydroxy esters and derivatives thereof

ABSTRACT

A process is provided for the preparation of a compound of formula (1) 
     
       
         
         
             
             
         
       
     
     wherein R and R′ represent optionally substituted hydrocarbyl groups and X represents a hydrocarbyl linking group. The process comprises either the stereoselective reduction of the keto group in a dihydroxy keto precursor followed by selective esterification of a primary hydroxy, or selective esterification of a primary hydroxy of a dihydroxy keto precursor followed by stereoselective reduction of the keto group.

The present invention concerns a stereoselective process for thepreparation of dihydroxy esters, and derivatives thereof.

According to a first aspect of the present invention, there is provideda process for the preparation of a compound of formula (1)

which comprises either a) the stereoselective reduction of a compound offormula (2)

to produce a compound of formula (3), and

-   b) Esterification of the compound of formula (3) in the presence of    a compound of formula R″—O—COR′ and a lipase or hydrolase enzyme    thereby to form the compound of formula (1); or-   c) Esterification of a compound of formula (2) in the presence of a    compound of formula R″—O—COR′ and a lipase or hydrolase enzyme    thereby to form the compound of formula (4), and

-   d) the stereoselective reduction of a compound of formula (4) to    produce a compound of formula (1)    wherein    X represents an optionally substituted hydrocarbyl linking group    R and R″ each independently represent an optionally substituted    hydrocarbyl group, and    R′ represents an optionally substituted hydrocarbyl, preferably an    optionally substituted alkyl group.

Hydrocarbyl groups represented by X, R, R′ or R″ may be substituted byone or more substituents, and may be per-substituted, for exampleperhalogenated. Examples of substituents include halo, especially fluoroand chloro, alkoxy, such as C₁₋₄alkoxy, and oxo.

Preferably, X represents a group of formula —(CH₂)_(n)— where n is from1 to 4, and most preferably X represents a group of formula —CH₂—.

R″ may be an alkyl group, such as a C₁₋₆ alkyl group, or analkylcarbonyl group, such as a C₁₋₆alkylcarbonyl group, for example aCH₃(C═O)— or CF₃(C═O)— group. R″ is most preferably a vinyl orisopropenyl group.

R preferably represents a C₁₋₆ alkyl group, which may be linear orbranched, and may be substituted by one or more substituents. Mostpreferably, R represents a t-butyl group.

R′ may represent a substituted alkyl, often C₁₋₆ alkyl group, such as aCF₃— or CF₃CH₂— group, but is preferably an unsubstituted C₁₋₆ alkylgroup, and most especially a methyl group.

The stereoselective reduction of the compounds of formulae (2) or (4)preferably employ chemical or microbial reduction methods, such ashydrogenation, transfer hydrogenation, metal hydride reduction ordehydrogenases. Examples of a suitable hydrogenation process such asthat described in Helv. Chim. Acta 69, 803, 1986 (incorporated herein byreference) include the use of between 0.01 and 10% (w/w) of catalystssuch as platinum, palladium or rhodium on heterogeneous supports such ascarbon, alumina, silica using molecular hydrogen at between 1 and 10 barin a solvent such as methanol, ethanol, t-butanol, dimethylformamide,t-butylmethylether, toluene or hexane. Alternatively homogenoushydrogenation catalysts such as those described in EP0583171(incorporated herein by reference) may be used.

Examples of suitable chemical transfer hydrogenation processes includethose described in Zassinovich, Mestroni and Gladiali, Chem. Rev. 1992,92, 1051 (incorporated herein by reference) or by Fuji et al in J. Am.Chem. Soc. 118, 2521, 1996 (incorporated herein by reference). Preferredchemical transfer hydrogenation processes employ chiral ligatedcomplexes of transition metals, such as ruthenium or rhodium, especiallychiral diamine-ligated neutral aromatic ruthenium complexes. Preferably,such a chemical transfer hydrogenation employs an acid, especially aformate salt such as triethylammonium formate, as the hydrogen source.

Metal hydride reagents such as those described in Tet. 1993, 1997, Tet.Asymm. 1990, 1, 307, (incorporated herein by reference), or J. Am. Chem.Soc. 1998, 110, 3560 (incorporated herein by reference) can be used.

Examples of suitable microbial reductions include contacting thecompound of formula (2) or (4) with an organism possessing theproperties of a microorganism selected from Beauveria preferablyBeauveria bassiana, Pichia preferably Pichia angusta or Pichia pastoris,trehalophila, haplophila or membranefaciens. Candida preferably Candidahumicola, solani, guillermondii, diddenssiae or friedrichii.Kluyveromyces preferably Kluyveromyces drosophilarum, or Torulasporapreferably Torulaspora hansenii. The reduction may be achieved bycontacting the compounds of formulae (2) or (4) with an enzyme extractedfrom the foregoing microorganisms. Most preferably, the compounds offormulae (2) or (4) are contacted with a microorganism selected fromPichia angusta, Pichia pastoris, Candida guillermondii, Saccharomycescarlsbergensis, Pichia trehalophila, Kluyveromyces drosopliarum andTorulospora hansenii, or an extract from the foregoing organisms.

The invention preferably comprises producing a compound of formula (3)by selectively reducing a compound of formulae (2) using whole cells ofor extracts from the aforementioned microorganisms, preferably Pichiaangusta, Pichia pastoris, Candida guillermondii, Saccharomycescarlsbergensis, Pichia trehalophila, Kluyveromyces drosopliarum andTorulospora hansenii.

The invention is most preferably carried out using whole cells of theorganisms as this avoids the need to separate the desired enzyme andprovides co-factors for the reaction.

Any of the above species may be used but in many embodiments, it hasbeen found that high conversions and high selectivity can be achieved bythe use of the enzyme or whole cells of Pichia angusta.

In general a co-factor, normally NAD(P)H (nicotinamide adeninedinucleotide or nicotinamide adenine dinucleotide phosphate) and asystem for re-generating the co-factor, for example glucose and glucosedehydrogenase, are used with the enzymes to drive the reaction. Assuitable co-factors and reduction mechanisms are present in the wholecells it is preferred to use the whole cells in a nutrient medium whichpreferably contains a suitable carbon source, which may include one ormore of the following: a sugar, e.g. maltose, sucrose or preferablyglucose, a polyol e.g. glycerol or sorbitol, citric acid, or a loweralcohol, for example methanol or ethanol.

If whole cells are intended to grow during the reaction nitrogen andphosphorus sources and trace elements should be present in the medium.These may be those normally used in culturing the organism.

The process may be carried out by adding a compound of formula (2) or(4) to a culture of the growing organism in a medium capable ofsupporting growth or to a suspension of the live cells in a medium whichpreferably contains a carbon source but which lacks one or morenutrients necessary for growth. Dead cells may also be used providingthe necessary enzymes and co-factors are present; if necessary they maybe added to the dead cells.

If desired the cells may be immobilised on a support which is contactedwith compound of formula (2) or (4) preferably in the presence of asuitable carbon source as previously described.

The pH is suitably 3.5 to 9, for example 4 to 9, preferably at most 6.5and more preferably at most 5.5. Very suitably a pH of 4 to 5 is used.The process may suitably be carried out at a temperature of 10 to 50°C., preferably 20 to 40° C. and more preferably 25 to 35° C. It ispreferred to operate under aerobic conditions if live whole cells of theaforesaid organisms are present. An aeration rate equivalent to 0.01 to1.0 volumes of air measured at standard temperature and pressure pervolume of the culture medium per minute is suitably employed at theaforesaid conditions of pH and temperature but it will be appreciatedthat considerable variation is possible. Similar pH, temperature andaeration conditions may be used during growth of the organisms if thisis carried out separately from the process.

Purified enzymes may be isolated by known means suitably by centrifuginga suspension of disintegrated cells and separating a clear solution fromdebris, separating the desired enzyme from the solution for example byion exchange chromatography suitably with elution from the column withliquid of increasing ionic strength and/or by selective precipitation bythe addition of an ionic material, for example ammonium sulphate. Suchoperations may be repeated if desired to enhance purity.

The microbial reduction of compounds of formula (2) or (4) isparticularly preferred, and this process forms a second aspect of thepresent invention.

In the esterification of compounds of formula (2) or (3) it is preferredto transesterify with another ester, which is present in at least moleequivalence with respect to the alcohol and is suitably a vinyl ester(as the by-product, acetaldehyde is not involved in a back-reaction).Alternatively an anhydride such as acetic anhydride or trifluoroaceticanhydride, or an ester such as ethylacetate or a fluorinated ester suchas trifluoroethylacetate may be used. It is preferred that theregiospecific esterification reaction be carried out in an organicsolvent containing less than 1% (w/w) water such as acetonitrile,ethylacetate, tetrahydrofuran, tert-butylmethylether, toluene, butanone,pentanone or hexanone at a temperature of preferably 20 to 75° C., morepreferably 25 to 50° C. The esters are preferably esters of loweralkanoic acids having 2 to 8 carbon atoms, or substituted derivativesthereof. Optionally an inert atmosphere may be employed, for example aflow of nitrogen may be passed through the solution.

The enzymes may be provided as such or as whole cells comprising them.It is preferred that they be immobilised so as to facilitate theirseparation from the product and, if desired, re-use.

Preferred enzymes include lipases such as Porcine pancreatic lipase.Candida cylindracea lipase. Pseudomonas fluorescens lipase. Candidaantarctica fraction B such as that available under the trade markChirazyme L2, those from Humicola lanuginosa for example that sold underthe Trade Mark Lipolase or those from Pseudomonas for example that soldunder the Trade Mark SAM II and more preferably those from Candidaantarctica, for example that sold under the Trade Mark Chirazyme.

Compounds of formula (1) wherein R′ is CH₃, R is optionally substitutedhydrocarbyl. X is —(CH₂)_(n)— and n is 1 to 4 form a third aspect of thepresent invention. Preferably. R is t-butyl and most preferably, X is—CH₂—.

Compounds of formula (1) are useful intermediates for the preparation ofpharmaceutical compounds. Commonly, they are reacted with a protectinggroup for 1,3-dihydroxy moieties such as 2,2-dimethoxypropane to form anacetonide as described in Synthesis 1998, 1713. The group R′—(C═O)— maythen be selectively removed by treatment with weakly basic alcoholicsolution eg K₂CO₃ solution as described in U.S. Pat. No. 5,278,313 or alipase either in aqueous solution, or in organic solution containingsufficient water to support hydrolysis, to form a compound of formula(5):

This process for the preparation of compounds of formula (5) forms afourth aspect of the present invention.

EXAMPLE 1 Preparation of (3R,5S) t-butyl 3,5,6-trihydroxyhexanoate

To a stirred 250 ml round bottom flask 20 ml acetonitrile, 0.405 g(0.662 mmoles) of di-mu-chlorobis[(p-cymene)chlororuthenium (II)], and0.492 g (1.34 mmoles)(1S,2S)-(+)-N-(4-toluenesulfonyl)-1,2-diphenylethylenediamine werecharged. The solution was de-oxygenated by sparging with nitrogen andthereafter maintaining a trickle. A de-oxygenated solution of 26 g(0.119 mol) optically pure (5S) t-butyl 3-keto-5,6-dihydroxyhexanoate in15 m| acetonitrile was charged to the reaction vessel and the solutionstirred at ambient temperature for 20 minutes, 65 ml of a 5:2 (mol/mol)mixture of distilled formic acid and triethylamine were then added overa period of 10 minutes and the reaction mixture stirred at ambienttemperature for 48 hours. To this solution 80 ml dichloromethane and 120ml saturated sodium bicarbonate were slowly added. 70 g ammoniumchloride was charged to the aqueous layer and the organic layerseparated. The aqueous layer was washed thrice more with 90 mlethylacetate, the organic fractions combined, dried over sodium sulfateand the solvent removed to give 21.1 g of a crude oil containing mainly(3R,5S) t-butyl 3,5,6-trihydroxyhexanoate. The ratio of diastereomerswas determined by ¹³CNMR to be 5.2:1 (3R:5S): (3S:5S). The material wasused crude in the next reaction but could be purified by columnchromatography.

Preparation of (3R,5S) t-butyl 6-acetoxy-3,5-dihydroxyhexanoate

To a stirred 1 l round bottom flask 700 ml tetrahydrofuran and 70.7 g(0.32 mol) of (3R,5S) t-butyl 3,5,6-trihydroxyhexanoate, 41 ml (0.46mol) vinylacetate and 6.3 g of the supported lipase Chirazyme L2™ werecharged. After 3 hours stirring at ambient temperature the lipase wasremoved by screening and the volatiles removed by distillation undervacuum. The mass of crude oil was 78.7 g and the major component wasdetermined to be (3R,5S) t-butyl 6-acetoxy-3,5-dihydroxyhexanoate. Thismaterial was used directly in the next stage.

Preparation of(4R,6S)-6-[(acetyloxy)methyl]-2,2-dimethyl-1,3-dioxane-4-acetic acid,1,1-dimethylethylester

To a stirred 1 litre round bottom flask 78.7 g (3R,5S) t-butyl6-acetoxy-3,5-dihydroxyhexanoate, 800 ml of 2,2-dimethoxypropane and 5.7g p-toluenesulfonic acid were charged. After 35 minutes thee reactionmass was concentrated to half its volume and 300 ml of dichloromethaneand 300 ml of 1M sodiumbicarbonate added. The organic layer wasseparated and the aqueous layer washed thrice more with 150 mlethylacetate. The organic fractions were combined, dried over sodiumsulfate and the volatiles removed by distillation under vacuum. 92 g ofa crude oil was obtained. This was purified first by passing through ashort column of flash silica and eluting with hexane and thenhexane:ethylacetate 85:15 (v/v), and then crystallising the material 3times from hexane to give 22.17 g(4R,6S)-6-[(acetyloxy)methyl]-2,2-dimethyl-1,3-dioxane-4-acetic acid,1,1-dimethylethylester which by chiral GC was determined to be 99.9% de.

Preparation of(4R,6S)-6-(hydroxymethyl]-2,2-dimethyl-1,3-dioxane-4-acetic acid,1,1-dimethylethylester

To a 500 ml stirred round bottom flask 22.17 g of(4R,6S)-6-[(acetyloxy)methyl]-2,2-dimethyl-1,3-dioxane-4-acetic acid,1,1-dimethylethylester, 250 ml methanol and 5.05 g crushed potassiumcarbonate were charged. The reaction was stirred for 35 minutes untilthe hydrolysis was complete, then the potassium carbonate was removed byscreening, the reaction mass concentrated and 150 ml 5% (w/w) brine and150 ml toluene added. The organic layer was separated and the aqueouswashed twice more with 250 ml toluene. The organic layers were combined,washed three times with 15% (w/w) brine and the solvent removed byvacuum distillation to give 17.78 g of clear oil, which was determinedto be >99% (4R,6S)-6-(hydroxymethyl)-2,2-dimethyl-1,3-dioxane-4-aceticacid, 1,1-dimethylethylester.

EXAMPLE 2 Preparation of (5S) tert-butyl6-acetoxy-5-hydroxy-3-ketohexanoate

To a stirred 250 ml round bottom flask were charged 2.32 g (0.0106moles) (5S) tert-butyl 5,6-dihydroxy-3-ketohexanoate, 40 mltetrahydrofuran, 0.98 ml (0.0106 moles) vinyl acetate and 0.22 g of thesupported lipase Chirazyme L2™. After 20 minutes the lipase was removedby screening and the volatiles removed by distillation under vacuum, togive 2.96 g of a crude oil that was characterised by NMR as (5S)tert-butyl 6-acetoxy-5-hydroxy-3-ketohexanoate.

EXAMPLE 3 Preparation of (3R,5S) t-butyl 3,5,6-trihydroxyhexanoate

Pichia angusta NCYC R230 (deposited under the provisions of the BudapestTreaty on May 18^(th), 1995) was grown in a Braun Biostat Qmulti-fermenter system in the following medium (per litre): glucose 40g; MgSO₄, 1.2 g; K₂SO₄, 0.21 g; KH₂PO₄, 0.69 g; H₃PO₄ (concentrated), 1ml; yeast extract (Oxoid), 2 g; FeSO₄.7H₂O, 0.05 g; antifoam (EEA 142Foammaster), trace elements solution, 1 ml (this solution contained perlitre CuSO₄.5H₂O, 0.02; MnSO₄.4H₂O, 0.1 g; ZnSO₄.7H₂O, 0.1 g; CaCO₃, 1.8g.

Each of 4 fermenters were charged with 250 ml of medium and sterilisedby autoclaving. The pH was adjusted to 4.5 using 7 molar ammoniumhydroxide solution, the temperature was set to 28° C., the air flow setat 300 ml/minute and the agitator speed set to 1200 rpm. Fermenters wereinoculated with cells taken from agar plates (2% agar) comprising thesame medium as described above except that the glucose concentration was20 g/litre. Following 22 hours growth in the fermenters the bioreductionreaction was started by the addition of (5S) t-butyl3-keto-5,6-dihydroxyhexanoate; two of the fermenters were charged with3.75 ml each and the other two charged with 5 ml each.

The reaction was continued for a further 78 hours until 100% conversionof substrate. During this period the culture was fed with a 50% solutionof glucose at a rate of 1-3 grams glucose/litre culture/hour to maintaincell viability and provide a source of reducing power. Reactions wereterminated by removal of the cells by centrifugation. To the recoveredcell-free supernatant was added sodium chloride to a final concentrationof 20% w/v and the mixture extracted three times with an equal volume ofacetonitrile. The pooled acetonitrile extracts were dried with anhydroussodium sulfate and the solvent removed under reduced pressure in arotary evaporator (water bath temperature 45° C.) to yield a viscouspale yellow oil. The identity of the product from each reaction wasconfirmed as (3R,5S) t-butyl 3,5,6-trihydroxyhexanoate and thediastereomeric excess of each of the samples is given in the tablebelow.

diastereomeric experiment excess (%) 1 99.6 2 99.6 3 99.4 4 99.6

EXAMPLE 4 Preparation of (3R,5S) t-butyl 3,5,6-trihydroxyhexanoate

Pichia angusta NCYC R320 (deposited under the provisions of the BudapestTreaty on May 18, 1995) was grown in a Braun Biostat Q multi-fermentersystem in the following medium (containing, per litre): glucose, 20 g;ammonium sulfate, 10 g; Yeast extract (Oxoid), 2 g; MgSO₄.7H₂O, 1.2 g;KH₂PO₄, 0.69 g; K₂SO₄, 0.21 g; FeSO₄.7H₂O, 0.05 g; H₃PO₄ (concentrated),1 ml; EEA 142 “foammaster” antifoam, 0.5 ml; trace elements solution, 1ml (this solution contained per litre Ca(CH₃CO₂)₂, 2.85 g; ZnSO₄.7H₂O,0.1 g; MnSO₄.H₂O 0.075 g CuSO₄.5H₂O, 0.02 g; sulphuric acid(concentrated), 1 ml).

One fermenter was charged with 250 ml medium and sterilised byautoclaving. The pH was adjusted to 5.0 using 2 molar sodium hydroxidesolution. The temperature was set to 28° C., the air flow was set to 250ml minute⁻¹ and the agitator speed set to 1200 rpm. The fermenter wasinoculated with 2.5 ml of a suspension of cells in sterile deionisedwater prepared from an agar plate of Pichia angusta NCYC R320. Following17 hours growth the bioreduction was started by the addition of 6.36 g5(S) t-butyl 3-keto-5,6-dihydroxyhexanoate as an aqueous solution. Atthe same time a glucose feed to the fermenter was started at a rate of 2g glucose L⁻¹ h⁻¹.

The reaction was continued for a further 78 hours at which point 96%conversion of the substrate had been achieved. Starting material andproduct were detected by HPLC (Hichrom S5 CN-250A column, temperature35° C., mobile phase: aqueous TFA (0.1%): acetonitrile 95:5, flow rate 1ml min⁻¹, injection volume 5 ml, refractive index detector).

The reaction was terminated by the removal of cells by centrifuging at4000×g for 20 minutes. The pH of the recovered cell-free supernatant wasadjusted to 7.5 using 2M NaOH. MgSO₄.1.6H₂O (15% w/v based on anhydrous)was dissolved in the cell-free supernatant and the resulting solutionwas extracted twice with an equal volume of 2-pentanone. The solventphases were collected and the solvent removed under reduced pressure ina rotary evaporator at 45° C. yielding an orange viscous oil. This wasre-dissolved in 50 ml dry, distilled 2-pentanone and again the solventwas removed by rotary evaporation to afford t-butyl3,5,6-trihydroxyhexanoate (5.08 g, 80% isolated yield). Diastereomericexcess was determined as follows; a sample of t-butyl3,5,6-trihydroxyhexanoate (30 mg) was derivatised by reaction for atleast 10 minutes at room temperature in an excess of trifluoroaceticanhydride, excess anhydride was removed under a stream of dry nitrogenand the residual oil diluted with dichloromethane (1 ml). The sample wasanalysed using a Chiralcel Dex CB column (25 metre) at a temperature of140° C. (isothermal). The diastereomers eluted at 14.4 minutes (3R,5Sdiastereomer) and 15.7 minutes (3S,5S diastereomer). The diastereomericexcess of the sample was found by this method to be 99.7%.

1-16. (canceled)
 17. A process for the preparation of a compound offormula (4)

which comprises esterification of a compound of formula (2)

in the presence of a compound of formula R″—O—COR′ and a lipase orhydrolase enzyme thereby to form the compound of formula (4); wherein Xrepresents an optionally substituted hydrocarbyl linking group; R and R″each independently represent an optionally substituted hydrocarbylgroup; and R′ represents an optionally substituted hydrocarbyl group.18-20. (canceled)
 21. The process according to claim 17 wherein X is—CH₂—.
 22. The process according to claim 17 wherein R″ is a vinyl orisopropenyl group.
 23. The process according to claim 17 wherein R′represents a substituted or unsubstituted C₁₋₆ alkyl group.
 24. Theprocess according to claim 17 wherein R′ is CH₃, R is t-butyl and X is—CH₂—.
 25. The process according to claim 17 wherein the enzyme isselected from the group consisting of Porcine pancreatic lipase, Candidacylindracea lipase, Pseudomonas fluorescens lipase, Candida antarcticafraction B and lipase from Humicola lanuginosa.
 26. The processaccording to claim 17 wherein the compound of formula R″—O—COR′ is vinylacetate.